The invention relates to a process for achieving high, thermally induced expression rates, which last for a relatively long period of time, of proteins, e.g., enzymes, in recombinant host cells, in particular E. coli, by means of mutually suited cultivation parameters such as cell density, induction temperature and added amino-acid-containing substrates.
It is the aim of every genetic engineering process to obtain, in particular, maximum possible yields of recombinant product. To date, a two-phase process has proved to be successful for this purpose. First optimum growth of the cells is aimed at for this purpose, in order to achieve a large biomass and amount of plasmid. At the same time, care has to be taken that, while growth is good, the amount of waste product, e.g., acetate, accumulated is not too high. In this first phase more than a negligible production of the recombinant protein is undesired, since the cell growth is, in some cases, greatly reduced by excessive substrate consumption and/or by the overproduction of the recombinant protein which, in some cases, also has a toxic effect. For this reason it is important that an expression system which can be regulated is available. In such a system, the promoter is repressed during the growth phase of the cells so that there is little or no production of recombinant protein. In a second phase at a high cell density or large biomass, the promoter is derepressed and the expression is induced thereby. Numerous expression systems which can be regulated and in which the induction of protein expression is effected by a temperature shift or by chemical induction are known for microorganisms. One of the best known and most efficient promoters for E. coli is the lambda P.sub.L promoter which is, for example, described in EP-A-0,041,767. The CI857 repressor which blocks the promoter is thermally inactivated by a temperature shift to about 42.degree. C., and the transcription of the structural gene is thus made possible (phase 2). Many enzymes and other proteins have to date been expressed by employing such a two-phase standard procedure in E. coli, thus, for example, EcoRI restriction endonuclease (Bottermann et al., Biotechnol, Bioeng. 27 (1985), p. 1320), .alpha.-amylase (Reinikainen et al., Biotechnol. Lett. 10 (3) (1988), p. 149), mutarotase (EP-A-0,307,730) or glucose dehydrogenase (EP-A-0,290,768). Growth and expression do not necessarily have to be sequential in one reactor in this procedure, but may also take place in two separate reactors in series.
However, the mentioned procedure which has hitherto been used in practice and is also used, in modified form and using other promoter systems which can be regulated for other species of bacteria, such as Bacillus, often has disadvantages which, in specific cases, can make the process impracticable. Thus, a decrease, which is severe in some cases, in the growth rate of the recombinant host cells is observed in most cases when the number of plasmid replications increases and, connected thereto, the overexpression of protein is increased (for example, Bently and Kompala; Biotechn. Bioeng. 33 (1989) p. 49).
Furthermore, it is known that a derepression of the promoter, for example by a temperature shift, can lead to a severe restriction of growth and to high losses of plasmid (for example, Siegel and Ryu, Biotechn. Bioeng. 27 (1985), p. 28). Furthermore, noteworthy is the frequent observation (for example, Peretti and Bailey, Biotechn. Bioeng. 32 (1988), p. 418) that, immediately after promoter induction, there is initially a very high recombinant protein biosynthesis rate, although this is usually only short-lasting and then decreases more or less rapidly. All these effects cause the net synthesis rate to be limited by the decrease in growth rate after promoter induction has taken place. Thus, not only is the yield of recombinant protein, in absolute terms, often insufficient but also new recombinant host cells have to be continually cultivated since the old cells become unproductive after a relatively short time and, in most cases, can no longer be regenerated. This is an economic disadvantage.
Thus, the object was to eliminate the above-mentioned disadvantages in the production of proteins and enzymes with the aid of host cells containing recombinant promoters which can be regulated by optimizing the cultivation parameters and to make possible high recombinant protein biosynthesis rates for a long period of time.